Some studies about pre-service teachers’ anxiety and difficulties in teaching has been widely conducted, and the subjects were undergraduate students. However, none of the previous studies examined EFL in-service teachers of Teacher professional Education Programme which have different education background. Some of the subjects were from English Literature who did not get teaching practice programme when they were in undergraduate programme. Therfore, this research attempted to measure EFL in-service teachers’ anxiety level, the differences between EFL in-service teachers’ anxiety based on their educational background at Teacher Professional Education Programme, and also the factors caused the anxiety. This research applied both quantitative and qualitative research design. There were 38 EFL in-service teachers of Teacher Professional Education Programme who became the subjects of this research. However, to carry out the interview, only four subjects were choosen to be interviewed. The instruments that used in this research were questionnaire and interview. The researcher collected the data by sharing the questionnaire in form of Google Form online through WhatsApp. Then, the data interview was also collected online by using WhatsApp as well. To analyze the data, the researcher utilized SPSS 20 version for quantitative data. Then, data interviews were interpreted. The results of this research showed that EFL in-service teachers of Teacher Professional Education Programme had moderate anxiety level with the mean was 73.86. Then, it was found that there is a statistically significant difference level of anxiety between EFL in-service teachers who graduated from English Literature Department and those who graduated from English Education Department. It was significant at p = .001 level. Moreover, there were four major factors that caused the EFL in-service teachers anxiety. Those factors were fear of negative evaluaton, lack of teaching experiences, classroom management, and fear of making mistakes. In conclusion, It was suggested that EFL in-service teachers should be well-prepared before teaching practicum so that they will face less difficulties and anxiety.